Endoglucanases for treatment of cellulosic material

ABSTRACT

The present invention relates to production of fermentable sugars from lignocellulosic material by enzymatic conversion. The fermentable sugars are useful e.g. in the production of bioethanol. Novel polypeptides having endoglucanase activity, polynucleotides encoding them and vectors and host cells containing the polynucleotides are disclosed. A method for treating cellulosic material with the novel endoglucanase as well as uses of the enzymes and enzyme preparations and a method of preparing them are described.

RELATED APPLICATION DATA

This application claims the benefit of U.S. provisional application Ser.No. 61/651,256 filed May 24, 2012, hereby incorporated by referenceherein in its entirety.

The work leading to this invention has received funding from theEuropean Community's Seventh Framework Programme FP7/2007-2013 undergrant agreement no 239341.

FIELD OF THE INVENTION

The present invention relates to production of fermentable sugars fromlignocellulosic material by enzymatic conversion. The fermentable sugarsare useful e.g. in the production of bioethanol, or for other purposes.In particular the invention relates to novel polypeptides,polynucleotides encoding them, and to vectors and host cells containingthe polynucleotides. The invention is further directed to a method fortreating cellulosic material with fungal endoglucanase or an enzymepreparation containing said enzyme. Still further the invention isdirected to uses of the polypeptides or enzyme preparations containingsaid polypeptides and to a method of preparing them.

BACKGROUND OF THE INVENTION

Limited resources of fossil fuels, and increasing amounts of CO₂released from them and causing the greenhouse phenomenon have raised aneed for using biomass as a renewable and clean source of energy.Biomass resources can be broadly categorized as agricultural orforestry-based, including secondary sources derived from agro and woodindustries, waste sources and municipal solid wastes. One promising,alternative technology is the production of biofuels i.e. (bio)ethanolfrom lignocellulosic materials. In the transportation sector biofuelsare for the time being the only option, which could reduce the CO₂emissions by an order of magnitude. The ethanol can be used in existingvehicles and distribution systems and thus it does not require expensiveinfrastructure investments. Sugars derived from lignocellulosicrenewable raw materials can also be used as raw materials for a varietyof chemical products that can replace oil-based chemicals.

Lignocellulosic raw material comprises an abundant source ofcarbohydrates for a variety of biofuels, including bioethanol. Most ofthe carbohydrates in plants are in the form of lignocellulose, whichessentially consists of cellulose, hemicellulose, and lignin.Lignocellulose can be converted into bioethanol and other chemicalproducts via fermentation following hydrolysis to fermentable sugars. Ina conventional lignocellulose-to-ethanol process the lignocellulosicmaterial is first pretreated either chemically or physically to make thecellulose fraction more accessible to hydrolysis. The cellulose fractionis then hydrolysed to obtain sugars that can be fermented by yeast intoethanol and distilled to obtain pure ethanol. Lignin is obtained as amain co-product that may be used as a solid fuel.

One barrier of production of biofuels from cellulosic andlignocellulosic biomass is the robustness of the cell walls and thepresence of sugar monomers in the form of inaccessible polymers thatrequire a great amount of processing to make sugar monomers available tothe micro-organisms that are typically used to produce alcohol byfermentation. Enzymatic hydrolysis is considered the most promisingtechnology for converting cellulosic biomass into fermentable sugars.However, enzymatic hydrolysis is used only to a limited amount atindustrial scale, and especially when using strongly lignified materialsuch as wood or agricultural waste the technology is not satisfactory.The cost of the enzymatic step is one of the major economic factors ofthe process. Efforts have been made to improve the efficiency of theenzymatic hydrolysis of the cellulosic material (Badger 2002).

WO2001060752 describes a continuous process for converting solidlignocellulosic biomass into combustible fuel products. Afterpretreatment by wet oxidation or steam explosion the biomass ispartially separated into cellulose, hemicellulose and lignin, and isthen subjected to partial hydrolysis using one or more carbohydraseenzymes (EC 3.2).

WO2002024882 concerns a method of converting cellulose to glucose bytreating a pretreated lignocellulosic substrate with an enzyme mixturecomprising cellulase and a modified cellobiohydrolase I (CBHI) obtainedby inactivating its cellulose binding domain (CBD).

US 20040005674 A1 describes novel enzyme mixtures that can be useddirectly on lignocellulose substrate, whereby toxic waste productsformed during pretreatment processes may be avoided, and energy may besaved. The synergistic enzyme mixture contains a cellulase and anauxiliary enzyme such as xylanase, ligninase, amylase, protease,lipidase or glucuronidase, or any combination thereof. Cellulase isconsidered to include endoglucanase, beta-glucosidase andcellobiohydrolase. US 20050164355 describes a method for degradinglignocellulosic material with one or more cellulolytic enzymes selectedfrom endoglucanase, beta-glucosidase and cellobiohydrolase and in thepresence of at least one surfactant. Additional enzymes such ashemicellulases, esterase, peroxidase, protease, laccase or mixturethereof may also be used. The presence of surfactant increases thedegradation of lignocellulosic material compared to the absence ofsurfactant.

WO2011080317 describes a method of treating cellulosic material withfungal CBHII/Cel6A cellobiohydrolase enzyme. The enzyme is useful invarious industrial applications, particularly in production of biofuels,where production of fermentable sugars from lignocellulosic material atmoderate to elevated temperature is advantageous.

Cellulases from a number of bacterial and fungal sources have beenpurified and characterized. The best investigated and most widelyapplied cellulolytic enzymes of fungal origin have been derived fromTrichoderma reesei (the anamorph of Hypocrea jecorina). Cellulases fromless known fungi have also been disclosed. Hong et al. (2003a and 2003b)characterize EG and CBHI of Thermoascus aurantiacus produced in yeast.Tuohy et al. (2002) describe three forms of cellobiohydrolases fromTalaromyces emersonii, a moderately thermophilic fungus. The sequenceand detailed biochemical characterization of these T. emersoniicellobiohydrolases have shown comparable properties with thecellobiohydrolases of T. reesei and P. chrysosporium. The cellulaseenzymes of another thermophilic fungus, Melanocarpus albomyces, includeat least two endoglucanases (Cel45A and Cel7A) and one cellobiohydrolase(Cel7B). These enzymes have been cloned and characterized for their pHand temperature behavior (Miettinen-Oinonen et al., 2004). WO2007071818describes enzymatic conversion of lignocellulosic material by enzymesincluding cellobiohydrolase, endoglucanase, beta-glucosidase andoptionally xylanase derived from Thermoascus auranticus, Acremoniumthermophilium or Chaetomium thermophilium. U.S. Pat. No. 7,892,812describes cellulose compositions comprising endoglucanase and their usein industrial applications, for example in saccharification oflignocellulose biomass. The cellulases are from fungi Chrysosporiumlucknowense, which has been identified as Myceliophthora thermophila(Visser et al., 2011).

Endoglucanases of the Cel7 family (EGs fam 7) are disclosed e.g. in U.S.Pat. No. 5,912,157, which pertains Myceliphthora endoglucanase and itshomologues and applications thereof in detergent, textile, and pulp.U.S. Pat. No. 6,071,735 describes cellulases exhibiting highendoglucanase activity in alkaline conditions. Uses as detergent, inpulp and paper, and textile applications are discussed. U.S. Pat. No.5,763,254 discloses enzymes from strains of Humicola, Fusarium andMyceliopthora degrading cellulose/hemicellulose and having acarbohydrate binding module homologous to the region A of T. reesei.

WO2004078919 discloses purified glycosyl hydrolase family 7 (Cel7A)enzymes from Penicillium funiculosum, which demonstrate a high level ofspecific performance when formulated with an endoglucanase and tested onpretreated corn stover.

Haakana et al., (2004) describes the cloning and sequencing of threegenes encoding cellulases Cel45A, Cel7A and Cel7B from Melanocarpusalbomyces. These cellulases work well in biostoning, with lowerbackstaining compared to T. reesei. WO9714804 discloses Cel7A familyenzymes from Melanocarpus albomyces and its applications in textile anddetergent industry. Voutilainen et al., (2008) describes novel GH7family cellobiohydrolases from the thermophilic fungi Acremoniumthermophilum, Thermoascus auranticus and Chaetomium thermophilum activeon insoluble polymeric substrates and participating in the rate limitingstep in the hydrolysis of cellulose.

U.S. Pat. No. 5,393,670 describes the DNA, vectors and transformed hostencoding Trichoderma reesei endoglucanase I.

There is a continuous need for new methods of degrading cellulosicsubstrates, in particular lignocellulosic substrates, and for newenzymes and enzyme mixtures, which enhance the efficiency of thedegradation. There is also a need for enzymes and processes, which areversatile and which work not only at moderate temperatures but also athigh temperatures, thus increasing the reaction rates and enabling theuse of high biomass consistency leading to high sugar and ethanolconcentrations. This approach may lead to significant savings in energyand investment costs. The high temperature also decreases the risk ofcontamination during hydrolysis. The present invention aims to meet atleast part of these needs.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide novel polypeptideshaving endoglucanase activity and polynucleotides encoding thepolypeptides. The novel polypeptides may have improved specific activityand/or improved thermostability. The novel polypeptides may also haveversatile applications. A further object of the present invention is toprovide new enzymes and enzyme preparations, which enhance theefficiency of the cellulosic degradation. Especially the object of theinvention is to provide new enzymes having endoglucanase activity.Another object of the present invention is to provide a method fortreating cellulose material with an improved enzyme or enzymepreparation.

The objects of the invention are achieved by novel polypeptides of GHfamily 7 (Cel7) obtained from Acremonium thermophilium ALKO4245.

The present invention provides a polypeptide having endoglucanaseactivity and comprising an amino acid sequence having at least 57%identity to SEQ ID NO:7 (EG_A) or at least 58% identity to SEQ ID NO:8(EG_B) or a fragment or variant thereof having endoglucanase activity.

The invention further provides an isolated polynucleotide selected fromthe group consisting of:

a) a polynucleotide comprising the coding sequence as shown in SEQ IDNO: 5 or 6;

b) a polynucleotide encoding a polypeptide of claim 1;

c) a polynucleotide encoding a fragment of a polypeptide encoded by apolynucleotide of a) or b), wherein said fragment has endoglucanaseactivity; and

d) a polynucleotide comprising a nucleotide sequence which is degenerateto the nucleotide sequence of a polynucleotide of a) or b);

or the complementary strand of such a polynucleotide.

The invention is also directed to a vector, which comprises saidpolynucleotide and a host cell comprising said vector. Escherichia colistrains having accession number DSM 25492, DSM 25493, DSM 25657, DSM,25658, DSM 25655 and DSM 25656 are also included in the invention.

A further object of the invention is to provide a method of producingsaid polypeptide having endoglucanase activity, the method comprisingthe steps of transforming a host cell with an expression vector encodingsaid polypeptide, and culturing said host cell under conditions enablingexpression of said polypeptide, and optionally recovering and purifyingsaid polypeptide.

Other objects of the invention are the enzyme preparations comprising atleast one of the novel polypeptides and the use of said enzymepreparations and polypeptides in biofuel, biomass hydrolysis, starch,textile, detergent, pulp and paper, food, feed or beverage industry.

The invention also provides a method for treating cellulosic materialwith an endoglucanase or an enzyme preparation comprising saidendoglucanase, wherein the method comprises the following steps:

i) reacting the cellulosic material with said endoglucanase or theenzyme preparation comprising said endoglucanase

ii) obtaining at least partially hydrolyzed cellulosic material.

Specific embodiments of the invention are set forth in the dependentclaims. Other objects, details and advantages of the present inventionwill become apparent from the following drawings, detailed descriptionand examples.

The novel endoglucanase applicable in the method is capable ofhydrolysing cellulosic materials at moderate to elevated temperatures,particularly in combination with other enzymes used in hydrolysis ofcellulosic or lignocellulosic materials.

Endoglucanases obtainable from Acremonium thermophilum ALKO4245 areparticularly useful in hydrolysing and degrading cellulosic material.The enzymes are kinetically very effective over a broad range oftemperatures, and although they have high activity at standardhydrolysis temperatures, they are also very efficient at hightemperatures. This makes them extremely well suited for varyingcellulosic substrate hydrolysis processes carried out both atconventional temperatures and at elevated temperatures. In theconventional separate hydrolysis and fermentation process (SHF) thetemperature of enzymatic hydrolysis is typically higher than that offermentation. The use of thermostable enzymes in the hydrolysis offerpotential benefits, such as higher reaction rates at elevatedtemperatures, reduction of enzyme load due to higher specific activityand stability of enzymes, increased flexibility with respect to processconfiguration and decreased contamination risk. The general robustnessof thermostable enzymes compared to mesophilic ones also increases therecyclability of enzymes in the industrial process.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention will be described in greater detail bymeans of preferred embodiments with reference to the attached drawings,in which

FIG. 1 schematically shows the expression cassettes used in thetransformation of Trichoderma reesei protoplasts for overproducing therecombinant Acremonium thermophilum ALKO4245 EG/Cel7 proteins (EG_A andEG_B), Acremonium thermophilum ALKO4245 EG/Cel7+Trichoderma reeseiEGI/Cel7B_linker-CBM (EG_A+EGI-CBM and EG_B+EGI-CBM) and Acremoniumthermophilum ALKO4245 EG/Cel7+Trichoderma reesei CBHI/Cel7A_linker-CBM(EG_A+CBHI-CBM and EG_B+CBHI-CBM) fusion proteins. The Acremoniumthermophilum ALKO4245 cel7/egl genes (egl_A and egl_B) and cel7/egl-CBMfusion genes Acremonium thermophilum ALKO4245 cel7/egl+Trichodermareesei cel7B/egl1_linker-CBM (egl_A+egl1-CBM and egl_B+egl1-CBM) andAcremonium thermophilum ALKO4245 cel7/egl+Trichoderma reeseicel7A/cbh1_linker-CBM (egl_A+cbh1-CBM and egl_B+cbh1-CBM) were under thecontrol of T. reesei cel7A/cbh1 promoter (p cbh1) and the termination ofthe transcription was ensured by using T. reesei cel7A/cbh1 terminatorsequence (t cbh1). The amdS gene was included as a transformationmarker.

FIGS. 2A-2B show results from hydrolysis of steam exploded corn fibreperformed with enzyme mixtures comprising the EG/Cel7 endoglucanases ofthe invention. The corn fibre substrate was hydrolyzed using differentenzyme mixtures at a dosage of 0.5 mg of protein per g of total solidsall at 37° C. and 55° C. in high dry matter conditions. The compositionsof the enzyme mixtures; basis enzyme mixture (MIXTURE 1) andcompositions comprising the EG_A and EG_B, are described in more detailin Example 5. Samples from five different tubes were taken after 48hours hydrolysis time and quantified by HPLC, in which the concentrationof glucose was determined. The concentration of glucose is presented.

FIG. 2A shows the hydrolysis results of steam exploded corn fibreperformed at 37° C. with a basis enzyme mixture (MIXTURE 1) supplementedwith the EG_A (MIXTURE 1_EG_A) or EG_B (MIXTURE 1_EG_B).

FIG. 2B shows the hydrolysis results of steam exploded corn fibreperformed at 55° C. with a basis enzyme mixture (MIXTURE 1) supplementedwith the EG_A (MIXTURE 1_EG_A).

FIGS. 3A-3C show results from hydrolysis of steam exploded corn fibreperformed with enzyme mixtures comprising the EG_A+EGI-CBM andEG_B+EGI-CBM fusion protein of the invention. The corn fibre substratewas hydrolyzed using different enzyme mixtures at a dosage of 0.5 mg ofprotein per g of total solids all at 37° C. with both low and high drymatter conditions and at 55° C. with high dry matter conditions. Thecompositions of the enzyme mixtures, a basis enzyme mixture (MIXTURE 1)and compositions comprising the EG_A+EGI-CBM and EG_B+EGI-CBM, aredescribed in more detail in Example 5. Samples from five different tubeswere taken after 48 hours hydrolysis time and quantified by HPLC, inwhich the concentration of glucose was determined. The concentration ofglucose is presented.

FIG. 3A shows the hydrolysis results of steam exploded corn fibreperformed at 37° C. with low dry matter conditions and with a basisenzyme mixture (MIXTURE 1) supplemented with the EG_A+EGI-CBM orEG_B+EGI-CBM.

FIG. 3B shows the hydrolysis results of steam exploded corn fibreperformed at 37° C. with high dry matter conditions and with a basisenzyme mixture (MIXTURE 1) supplemented with the EG_B+EGI-CBM.

FIG. 3C shows the hydrolysis results of steam exploded corn fibreperformed at 55° C. with high dry matter conditions and with a basisenzyme mixture (MIXTURE 1) supplemented with the EG_B+EGI-CBM.

FIGS. 4A-4C show results from hydrolysis of steam exploded corn fibreperformed with enzyme mixtures comprising the EG_A+CBHI-CBM andEG_B+CBHI-CBM fusion protein of the invention. The corn fibre substratewas hydrolyzed using different enzyme mixtures at a dosage of 0.5 mg ofprotein per g of total solids all at 37° C. with both low and high drymatter conditions and at 55° C. with high dry matter conditions. Thecompositions of the enzyme mixtures; a basis enzyme mixture (MIXTURE 1)and compositions comprising the EG_A+CBHI-CBM and EG_B+CBHI-CBM, aredescribed in more detail in Example 5. Samples from five different tubeswere taken after 48 hours hydrolysis time and quantified by HPLC, inwhich the concentration of glucose was determined. The concentration ofglucose is presented.

FIG. 4A shows the hydrolysis results of steam exploded corn fibreperformed at 37° C. with low dry matter conditions and with a basisenzyme mixture (MIXTURE 1) supplemented with the EG_A+CBHI-CBM orEG_B+CBHI-CBM.

FIG. 4B shows the hydrolysis results of steam exploded corn fibreperformed at 37° C. with high dry matter conditions and with a basisenzyme mixture (MIXTURE 1) supplemented with the EG_A+CBHI-CBM orEG_B+CBHI-CBM.

FIG. 4C shows the hydrolysis results of steam exploded corn fibreperformed at 55° C. with high dry matter conditions and with a basisenzyme mixture (MIXTURE 1) supplemented with the EG_B+CBHI-CBM.

SEQUENCE LISTING

SEQ ID NO: 1 Sequence of the oligonucleotide primer egl9

SEQ ID NO: 2 Sequence of the oligonucleotide primer egl11 SEQ ID NO: 3Sequence of the PCR fragment obtained from Acremonium thermophilumALKO4245 (CBS 116240) using the primers egl9 and egl11.

SEQ ID NO: 4 Sequence of the PCR fragment obtained from Acremoniumthermophilum ALKO4245 (CBS 116240) using primers egl9 and egl11.

SEQ ID NO: 5 The nucleotide sequence of the Acremonium thermophilumALKO4245 (CBS 116240) egl_A gene.

SEQ ID NO: 6 The nucleotide sequence of the Acremonium thermophilumALKO4245 (CBS 116240) egl_B gene.

SEQ ID NO: 7 The deduced amino acid sequence of the Acremoniumthermophilum ALKO4245 (CBS 116240) EG_A.

SEQ ID NO: 8 The deduced amino acid sequence of the Acremoniumthermophilum ALKO4245 (CBS 116240) EG_B.

SEQ ID NO: 9 Sequence of the oligonucleotide primer egl50

SEQ ID NO: 10 Sequence of the oligonucleotide primer CBM_(—)1

SEQ ID NO: 11 Sequence of the oligonucleotide primer CBM_(—)2

SEQ ID NO: 12 Sequence of the oligonucleotide primer CBM_(—)17

SEQ ID NO: 13 Sequence of the oligonucleotide primer egl64

SEQ ID NO: 14 Sequence of the oligonucleotide primer CBM_(—)4

SEQ ID NO: 15 Sequence of the oligonucleotide primer CBM_(—)5

SEQ ID NO: 16 Sequence of the oligonucleotide primer CBM_(—)18

SEQ ID NO: 17 Sequence of the PCR fragment obtained from a plasmidcontaining the full-length Acremonium thermophilum ALKO4245 egl_A geneusing primers egl50 and CBM_(—)1.

SEQ ID NO: 18 Sequence of the PCR fragment obtained from a plasmidcontaining the full-length Acremonium thermophilum ALKO4245 egl_B geneusing primers egl64 and CBM_(—)4.

SEQ ID NO: 19 Sequence of the PCR fragment obtained from plasmidcontaining the Trichoderma reesei egl1 gene using primers CBM_(—)2 andCBM_(—)17.

SEQ ID NO: 20 Sequence of the PCR fragment obtained from plasmidcontaining the Trichoderma reesei egl1 gene using primers CBM_(—)5 andCBM_(—)18.

SEQ ID NO: 21 The nucleotide sequence of the Acremonium thermophilumALKO4245 (CBS 116240) egl_A+Trichoderma reesei egl1-CBM fusion gene.

SEQ ID NO: 22 The nucleotide sequence of the Acremonium thermophilumALKO4245 (CBS 116240) egl_B+Trichoderma reesei egl1-CBM fusion gene.

SEQ ID NO: 23 The deduced amino acid sequence of the Acremoniumthermophilum ALKO4245 (CBS 116240) EGA+Trichoderma reesei EGI-CBM fusionprotein.

SEQ ID NO: 24 The deduced amino acid sequence of the Acremoniumthermophilum ALKO4245 (CBS 116240) EG_B+Trichoderma reesei EGI-CBMfusion protein.

SEQ ID NO: 25 The nucleotide sequence of the Acremonium thermophilumALKO4245 (CBS 116240) egl_A+Trichoderma reesei cbh1-CBM fusion gene.

SEQ ID NO: 26 The nucleotide sequence of the Acremonium thermophilumALKO4245 (CBS 116240) egl_B+Trichoderma reesei cbh1-CBM fusion gene.

SEQ ID NO: 27 The deduced amino acid sequence of the Acremoniumthermophilum ALKO4245 (CBS 116240) EGA+Trichoderma reesei CBHI-CBMfusion protein.

SEQ ID NO: 28 The deduced amino acid sequence of the Acremoniumthermophilum ALKO4245 (CBS 116240) EG_B+Trichoderma reesei CBHI-CBMfusion protein.

DETAILED DESCRIPTION OF THE INVENTION

Cellulose is the major structural component of higher plants. Itprovides plant cells with high tensile strength helping them to resistmechanical stress and osmotic pressure. Cellulose is a β-1,4-glucancomposed of linear chains of glucose residues joined by β-1,4-glycosidiclinkages. Cellobiose is the smallest repeating unit of cellulose. Incell walls cellulose is packed in variously oriented sheets, which areembedded in a matrix of hemicellulose and lignin. Hemicellulose is aheterogeneous group of carbohydrate polymers containing mainly differentglucans, xylans and mannans. Hemicellulose consists of a linear backbonewith β-1,4-linked residues substituted with short side chains usuallycontaining acetyl, glucuronyl, arabinosyl and galactosyl. Hemicellulosecan be chemically cross-linked to lignin. Lignin is a complexcross-linked polymer of variously substituted p-hydroxyphenylpropaneunits that provides strength to the cell wall to withstand mechanicalstress, and it also protects cellulose from enzymatic hydrolysis.

“Cellulose” or “cellulosic material” as used herein, relates to anymaterial comprising cellulose, hemicellulose and/or lignocellulose as asignificant component. Examples of cellulosic material include textilefibers derived e.g. from cotton, flax, hemp, jute and the man-madecellulosic fibers as modal, viscose and lyocel.

“Lignocellulose” is a combination of cellulose and hemicellulose andpolymers of phenol propanol units and lignin. It is physically hard,dense, and inaccessible and the most abundant biochemical material inthe biosphere. “Lignocellulosic material” means any material comprisinglignocellulose. Such materials are for example: hardwood and softwoodchips, wood pulp, sawdust and forestry and wood industrial waste;agricultural biomass as cereal straws, sugar beet pulp, corn fibre, cornstover and corn cobs, sugar cane bagasse, stems, leaves, hulls, husks,and the like; waste products as municipal solid waste, newspaper andwaste office paper, milling waste of e.g. grains; dedicated energy crops(e.g., willow, poplar, swithcgrass or reed canarygrass, and the like).Preferred examples are corn stover, switchgrass, cereal straw, sugarcanebagasse and wood derived materials.

Cellulosic material is degraded in nature by a number of variousorganisms including bacteria and fungi which produce enzymes capable ofhydrolyzing carbohydrate polymers. Degradation usually requiresdifferent cellulases acting sequentially or simultaneously. Degradationof more complex cellulose containing substrates requires a broad rangeof various enzymes. For example hemicellulose is degraded byhemicellulases, like xylanases and mannanases. Hemicellulase is anenzyme hydrolysing hemicellulose.

“Cellulolytic enzymes” are enzymes having “cellulolytic activity”, whichmeans that they are capable of hydrolysing cellulosic substrates orderivatives thereof into smaller saccharides. Cellulolytic enzymes thusinclude both cellulases and hemicellulases. Cellulases as used hereininclude (1) endoglucanases (EG, EC 3.2.1.4) which cut internalbeta-1,4-glucosidic bonds; (2) exoglucanases or cellobiohydrolases (CBH,EC 3.2.1.91) that cut the di-saccharide cellobiose from the reducing ornon-reducing end of the crystalline cellulose polymer chain; (3)beta-1,4-glucosidases (BG, EC 3.2.1.21) which hydrolyze the cellobioseand other short cellooligosaccharides to glucose. The CAZY (carbohydrateactive enzymes) classification system collates glycosyl hydrolase (GH)enzymes into families according to sequence similarity, which have beenshown to reflect shared structural features. In addition to thiscellulases can be classified to various glycosyl hydrolase familiesaccording their primary sequence, supported by analysis of the threedimensional structure of some members of the family (Henrissat 1991,Henrissat and Bairoch 1993, 1996).

T. reesei has a well-known and effective cellulase system containing twoCBH's, two major and several minor EG's and several BG's. T. reesei CBHI(Cel7A) cuts sugar from the reducing end of the cellulose chain, has aC-terminal cellulose binding module (CBM) and may constitute up to 60%of the total secreted protein. T. reesei CBHII (Cel6A) cuts sugar fromthe non-reducing end of the cellulose chain, has an N-terminal cellulosebinding module and may constitute up to 20% of the total secretedprotein. Endoglucanases EGI (Cel7B), and EGV (Cel45A) have a cellulosebinding module (CBM) in their C-terminus, EGII (Cel5A) has an N-terminalCBM and EGIII (Cel12A) does not have a cellulose binding module at all.CBHI, CBHII, EGI and EGII are so called “major cellulases” ofTrichoderma comprising together 80-90% of total secreted proteins. It isknown to a man skilled in the art that an enzyme may be active onseveral substrates and enzymatic activities can be measured usingdifferent substrates, methods and conditions. Identifying differentcellulolytic activities is discussed for example in van Tilbeurgh et al.1988.

Many fungal hydrolases are modular proteins, and all of them contain acatalytic domain (CD)/core expressing cellulolytic activity. In additionto the CD, hydrolases may contain a carbohydrate binding module, alsonamed as cellulose binding domain (CBD), which can be located either atthe N- or C-terminus of the catalytic domain. CBM mediates the bindingof the cellulase to crystalline cellulose but has little or no effect oncellulase hydrolytic activity of the enzyme on soluble substrates. Thesetwo domains are typically connected via a flexible and highlyglycosylated linker region.

Glycoside hydrolase family 7 (GH7) comprises enzymes with several knownactivities, especially endoglucanase and cellobiohydrolase.“Endoglucanases (EG)” are enzymes that cut internal glycosidic bonds ofthe cellulose chain. They are 1,4-beta-D-glucan 4-glucanohydrolases andcatalyze endohydrolysis of 1,4-beta-D-glycosidic linkages in polymers ofglucose such as cellulose and derivatives thereof. Some endoglucanaseshave a naturally occurring cellulose binding domain, while others donot. Some endoglucanases have also xylanase activity (Bailey et al.,1993).

The present invention is based on studies, which attempted to find novelGH7 family endoglucanases which would improve hydrolysis efficiency ofcellulosic substrates and which could be used for versatileapplications. The identification of the novel enzymes was done usingknown molecular biology methods. The basic methods are described, forexample, in Sambrook and Russel, 2001. Two GH family 7 (Cel7)endoglucanases referred as EG_A and EG_B were obtained (Table 1).

TABLE 1 The EG/Cel7 endoglucanases of the invention nucleic acid aminoacid accession nr for Endoglucanase SEQ ID NO: SEQ ID NO: the depositionEG_A 5 7 25492 EG_B 6 8 25493

The novel EG/Cel7 endoglucanases according to the present invention areobtainable from Acremonium sp. preferably from Acremonium thermophiliumand more preferably from strain having the characteristics of strainALKO4245 deposited as CBS 116240. “Obtainable from” means that they canbe obtained from said species, but it does not exclude the possibilityof obtaining them from other sources. In other words they may originatefrom any organism including plants. Preferably they originate frommicroorganisms e.g. bacteria or fungi. The bacteria may be for examplefrom a genus selected from Bacillus, Azospirillum and Streptomyces. Morepreferably the enzyme originates from fungi (including filamentous fungiand yeasts), for example from a genus selected from the group consistingof Thermoascus, Acremonium, Chaetomium, Achaetomium, Thielavia,Aspergillus, Botrytis, Chrysosporium, Collybia, Fomes, Fusarium,Humicola, Hypocrea, Lentinus, Melanocarpus, Myceliophthora, Myriococcum,Neurospora, Penicillium, Phanerochaete, Phlebia, Pleurotus, Podospora,Polyporus, Rhizoctonia, Scytalidium, Pycnoporus, Talaromyces, Trametesand Trichoderma.

The novel EG/Cel7 polypeptides of the invention having endoglucanaseactivity preferably comprise an amino acid sequence having at least 57%identity to SEQ ID NO:7 (EG_A) or at least 58% identity to SEQ ID NO:8(EG_B) or a fragment or variant thereof having endoglucanase activity.According to one embodiment of the invention, the polypeptide has atleast 60, 65, 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 7or 8 or to its enzymatically active fragment. The EG/Cel7 polypeptideshaving endoglucanase activity are also herein simply calledendoglucanases.

By the term “identity” is here meant the global identity between twoamino acid sequences compared to each other from the first amino acidencoded by the corresponding gene to the last amino acid. The identityof the full-length sequences is measured by using EMBOSS NeedleNeedleman-Wunsch global alignment program at EBI (EuropeanBioinformatics Institute) http://www.ebi.ac.uk/Tools/psa/emboss_needle/with the following parameters: BLOSUM50, Gap open 10.0, Gap extend 0.5.The algorithm is described in Needleman and Wunsch (1970). The manskilled in the art is aware of the fact that results usingNeedleman-Wunsch algorithm are comparable only when aligningcorresponding domains of the sequence and using the same parameters ineach comparison. Consequently comparison of e.g. cellulase sequencesincluding CBM or signal sequences with sequences lacking those elementscannot be done.

By the term “fragment having endoglucanase activity” is meant anyfragment of a defined sequence that has endoglucanase activity. In otherwords a fragment having endoglucanase activity may be the mature proteinpart of the defined sequence, or it may be only a fragment of the matureprotein part, provided that it still has endoglucanase activity.

The novel polypeptides may also be variants of said polypeptides. A“variant” may be a polypeptide that occurs naturally e.g. as an allelicvariant within the same strain, species or genus, or it may have beengenerated by mutagenesis. It may comprise amino acid substitutions,deletions or insertions, but it still functions in a substantiallysimilar manner to the enzymes defined above i.e. it comprises a fragmenthaving endoglucanase activity.

The cellulolytic polypeptides are usually produced in the cell asprepolypeptides comprising a signal sequence that is cleaved off duringsecretion of the protein. They may also be further processed duringsecretion both at the N-terminal and/or C-terminal end to give a mature,enzymatically active protein. “A polypeptide having endoglucanaseactivity” thus denotes that the polypeptide may be either in immature ormature form, preferably it is in mature form, i.e. the processing hastaken place. In addition, the “mature form” means an enzyme which hasbeen cleaved from its carrier protein in fusion constructions.

The EG/Cel7 endoglucanases of the present invention are preferablyrecombinant enzymes, which may be produced in a generally known manner.A polynucleotide fragment comprising the endoglucanase gene is isolated,the gene is inserted under a strong promoter into an expression vector,the vector is transformed into suitable host cells and the host cellsare cultivated under conditions provoking production of the enzyme.Methods for protein production by recombinant technology in differenthost systems are well known in the art (Sambrook et al., 1989; Coen,2001; Gellissen, 2005). Preferably the enzymes are produced asextracellular enzymes that are secreted into the culture medium, fromwhich they can easily be recovered and isolated.

The recombinant polypeptide may be a fused polypeptide in which anotherpolypeptide is fused at the N-terminus or the C-terminus of thepolypeptide of the present invention. A fused polypeptide is produced byfusing a polynucleotide encoding another polypeptide to a polynucleotideof the present invention. Techniques for producing fusion polypeptidesare known in the art, and include ligating the coding sequences encodingthe polypeptides so that they are in frame and that expression of thefused polypeptide is under control of the same promoter(s) andterminator.

The polypeptide of the invention disclosed in SEQ ID NO: 7 naturallycontains a C-terminal CBM and a linker. “A linker” is a flexible andhighly glycosylated region which connects the catalytic domain and theCBM. As used herein the CBM includes also the linker region. In oneembodiment of the invention this native linker and CBM region may bereplaced by, e.g. a linker and a CBM from a Trichoderma or Chaetomiumspecies, preferably from Trichoderma reesei. In a preferred embodimentthe natural CBM of endoglucanase EG_A has been replaced with a CBM of T.reesei endoglucanase I (EGI/Cel7B) or a CBM of T. reeseicellobiohydrolase I (CHBI/Cel7A) and preferably the resulting fusionprotein comprises an amino acid sequence having SEQ ID NO: 23(EG_A+EGI-CBM) or SEQ ID NO:27 (EG_A+CHBI-CBM) (Table 2).

The polypeptide of the invention disclosed in SEQ ID NO: 8 does notnaturally contain a CBM and a linker. In one embodiment of the inventionthis polypeptide may be attached by, e.g. a linker and a CBM regionsfrom a Trichoderma or Chaetomium species, preferably from Trichodermareesei. In a preferred embodiment the linker and CBM of T. reeseiEGI/Cel7B or T. reesei CHBI/Cel7A has been genetically attached to theendoglucanase EG_B and preferably the resulting fusion protein comprisesan amino acid sequence having SEQ ID NO: 24 (EG_B+EGI-CBM) or SEQ ID NO:28 (EG_B+CHBI-CBM) (Table 2).

TABLE 2 EG + CBM recombinant fusion proteins of the invention accessionEG + CBM fusion nucleic acid amino acid nr for the protein SEQ ID NO:SEQ ID NO: deposition EG_A + EGI-CBM 21 23 DSM 25657 EG_B + EGI-CBM 2224 DSM 25658 EG_A + CBHI-CBM 25 27 DSM 25655 EG_B + CBHI-CBM 26 28 DSM25656

Further, within the scope of the invention are recombinant fusionproteins comprising an amino acid sequence having at least 55% sequenceidentity to SEQ ID NO: 23 (EG_A+EGI-CBM) or SEQ ID NO: 27(EG_A+CHBI-CBM), or at least 64% sequence identity to SEQ ID NO: 24(EG_B+EGI-CBM) or SEQ ID NO: 28 (EG_B+CHBI-CBM). According to oneembodiment of the invention the fusion protein comprises an amino acidsequence having at least 60, 65, 70, 75, 80, 85, 90, 95, 98 or 99%identity to SEQ ID NO: 23 or 27, or at least 65, 70, 75, 80, 85, 90, 95,98 or 99% identity SEQ ID NO: 24 or 28 or to its enzymatically activefragment.

The EG/Cel7 endoglucanases of the invention may be used without a signalsequence and/or CBM or the signal sequence and/or CBM may derive fromdifferent enzymes of the above mentioned microorganisms or differentmicroorganism or be synthetically or recombinantly incorporated to thecatalytic domain of the above enzymes.

The invention relates to novel polynucleotides which may comprise anucleotide sequence of SEQ ID NO: 5 or 6, or a sequence encoding a novelpolypeptide as defined above, including complementary strands thereof.“Polynucleotide” as used herein refers to both RNA and DNA, and it maybe single stranded or double stranded. Further the polynucleotide may bedegenerate as a result of the genetic code to any one of the sequencesas defined above. This means that different codons may code for the sameamino acid.

One embodiment of the invention is an EG/Cel7 endoglucanase which isencoded by a polynucleotide sequence included in SEQ ID NO: 21, 22, 25or 26.

The polynucleotide may also be a fragment of said polynucleotidescomprising at least 17 nucleotides, preferably at least 20, 30, 40 or 50nucleotides. According to one embodiment of the invention thepolynucleotide is having a sequence set forth as SEQ ID NO 1, 2, 9, 10,13 or 14.

According to another embodiment of the invention, the polynucleotidecomprises a gene similar to that included in a microorganism havingaccession number DSM 25492, DSM 25493, DSM 25657, DSM 25658, DSM 25655or DSM 25656 (Table 1, Table 2).

The EG/Cel7 endoglucanase of the invention may be produced from arecombinant expression “vector” comprising the nucleic acid molecule,which encodes the endoglucanase as characterized above, operably linkedto regulatory sequences capable of directing the expression of a geneencoding said endoglucanase in a suitable host. Said regulatorysequences may be homologous or heterologous to the production organismor they may originate from the organism, from which the gene encodingthe endoglucanase polypeptide of the invention is isolated. Theexpression vector may further comprise marker genes for selection of thetransformant strains or the selection marker may be introduced to thehost in another vector construct by co-transformation.

The production “host” can be any homologous or heterologous organismcapable of expressing the cellulolytic enzyme. Preferably the host is amicrobial cell, more preferably a fungus. Most preferably the host is afilamentous fungus. Preferred hosts for producing the cellulolyticenzymes are in particular strains from the genus Trichoderma orAspergillus. Preferably the recombinant host is modified to express andsecrete cellulolytic enzymes as its main activity or one of its mainactivities. This can be done by deleting genes encoding major homologoussecreted enzymes e.g. the four major cellulases of Trichoderma and byintegrating heterologous genes to a locus with high expression andproduction levels.

The present invention relates also to a method for producing apolypeptide having endoglucanase activity, said method comprising thesteps of transforming a host cell with an expression vector encodingsaid polypeptide, and culturing said host cell under conditions enablingexpression of said polypeptide, and optionally recovering and purifyingsaid polypeptide. The production medium may be a medium suitable forgrowing the host organism and containing inducers for efficientexpression.

The polypeptides of the present invention may be isolated, which in thepresent context may simply mean that the cells and cell debris have beenremoved from the culture medium containing the polypeptide. Convenientlythe polypeptides are isolated e.g. by adding anionic and/or cationicpolymers (flocculants) to the spent culture medium to enhanceprecipitation of cells and cell debris. The medium is then filtratedusing an inorganic filtering agent and a filter to remove theprecipitants formed. After this the filtrate is further processed usinga semipermeable membrane to remove excess of salts, sugars and metabolicproducts. The polypeptides can also be purified or concentrated bycrystallization.

The novel EG/Cel7 polypeptides obtained by the method of the presentinvention may be components of an enzyme preparation. The term “enzymepreparation” denotes to a composition comprising at least one of thenovel polypeptides described herein. The polypeptide in the enzymepreparation may be a recombinant protein having endoglucanase activityand comprising an amino acid sequence having at least 57% sequenceidentity to EG_A having SEQ ID NO: 7 or at least 58% sequence identityto EG_B having SEQ ID NO: 8. In one embodiment the enzyme preparationcomprises a polypeptide which is a recombinant fusion protein having atleast 55% sequence identity to SEQ ID NO: 23 (EG_A+EGI-CBM) or SEQ IDNO: 27 (EG_A+CHBI-CBM), or at least 64% sequence identity to SEQ ID NO:24 (EG_B+EGI-CBM) or SEQ ID NO: 28 (EG_B+CHBI-CBM). According to oneembodiment of the invention the enzyme preparation comprises apolypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 98 or 99%identity to SEQ ID NO: 23 or 27, or at least 65, 70, 75, 80, 85, 90, 95,98 or 99% identity SEQ ID NO: 24 or 28 or to its enzymatically activefragment. Preferably the enzyme preparation comprises at leastcellobiohydrolase, endoglucanase, beta-glucosidase and optionallyxylanase.

The enzyme preparation may also comprise at least one further enzymeselected from a group of cellobiohydrolase, endoglucanase,beta-glucosidase, beta-glucanase, xyloglucanase, xylanase,beta-xylosidase, mannanase, beta-mannosidase, α-glucuronidase, acetylxylan esterase, α-arabinofuranosidase, α-galactosidase, pectinase,involving endo- and exo-α-L-arabinases, endo- and exo-galactoronase,endopectinlyase, pectate lyase, and pectinesterase, phenol esterase,ligninase involving lignin peroxidase, manganese-dependent peroxidase,H₂O₂-generating enzyme and laccase with or without a mediator. Theenzyme preparation may contain any combination of these enzymes andEG/Cel7 endoglucanases of the invention, but the enzymes are not limitedto those described herein. They can for example also be commerciallyavailable enzyme preparations.

The enzyme preparation may be in the form of liquid, powder orgranulate. It may be a filtrate containing one or more cellulolyticenzymes. Preferably the enzyme preparation is a spent culture medium.“Spent culture medium” refers to the culture medium of the hostcomprising the produced enzymes. Preferably the host cells are separatedfrom the said medium after the production. The enzyme preparation orcomposition may also be a “whole culture broth” obtained, optionallyafter inactivating the production host(s) or microorganism(s) withoutany biomass separation, down-stream processing or purification of thedesired cellulolytic enzyme(s). In the consolidated bioprocess theenzyme composition or at least some of the enzymes of the enzymecomposition may be produced by the fermentative microorganism.

The enzyme preparation may contain the enzymes in at least partiallypurified and isolated form. It may even essentially consist of thedesired enzyme or enzymes. The culture medium with or without host cellsmay be utilized as an enzyme preparation as such without furtherpurification, because the endoglucanase proteins can be secreted intothe culture medium, and they display activity in the ambient conditionsof the spent culture medium.

In addition to the endoglucanase proteins, the enzyme preparation of theinvention may contain additives, such as mediators, stabilizers,buffers, preservatives, surfactants and/or culture medium components.Preferred additives are such, which are commonly used in enzymepreparations intended for a particular application.

In the method of the present application for treating cellulosicmaterial the cellulosic material is reacted with the EG/Cel7endoglucanase of the invention or the enzyme preparation comprising saidendoglucanase, whereby at least partially hydrolyzed cellulosic materialis obtained. The enzymes are added in an enzymatically effective amounteither simultaneously e.g. in the form of an enzyme mixture, orsequentially, or are produced by the fermented microorganisms or ascombination of these methods

The EG/Cel7 endoglucanases of the invention are capable of hydrolyzingcellulosic material at moderate to elevated temperatures. The term“moderate temperature” or “conventional temperature” in context of thepresent invention means temperatures commonly used in cellulosehydrolysis and corresponding to the optimal temperatures or thermalstabilities of the enzymes used in such processes. Thus, the terms referto temperature ranges from 30° C. to 45° C. The term “elevatedtemperature” or “high temperature” refers to temperature ranges from 45°C. to 70° C. In short term hydrolysis processes the enzymes may beeffective even up to 80° C. Enzymes active or stable at such elevatedtemperature ranges are also called “thermostable” or “thermophilic”enzymes. The endoglucanases of the invention are used preferably attemperatures between 35° C. and 60° C. More preferably they are used attemperatures between 37° C. and 55° C., most preferably at temperaturesbetween 45° C. and 55° C.

The EG/Cel7 endoglucanases of the invention show improved hydrolysisresults both at moderate and elevated temperatures when compared to theenzyme mixtures containing conventional T. reesei endoglucanaseEGI/Cel7B. Different enzyme mixtures and combinations may be used tosuit different process conditions. Elevated temperatures are known toenhance the hydrolysis of crystalline cellulose present in cellulosic orlignocellulosic materials, thus reducing the total amount of enzymesneeded in hydrolysis or reducing the required hydrolysis time. Also,since at elevated temperatures the viscosity of the lignocellulosicsubstrate is decreased, thermostable enzymes make it possible to work athigher solid loadings and save in investment costs.

Particularly improved results at elevated temperatures may be obtainedwhen using an enzyme preparation comprising recombinant endoglucanaseEG_A having at least 57% sequence identity to SEQ ID NO: 7. In onepreferred embodiment of the invention the enzyme preparation comprisesAcremonium thermophilum cellobiohydrolase CBHI/Cel7A, Acremoniumthermophilum cellobiohydrolase CBHII/Cel6A, Thermoascus aurantiacusendoglucanase EGII/Cel5A, Acremonium thermophilum beta-glucosidaseβG/Cel3A, Thermoascus aurantiacus xylanase Xyn10A and endoglucanase EG_Aof the present invention.

In one embodiment of the invention the enzyme preparation comprisesrecombinant EG_A or EG_B fusion proteins having SEQ ID NOs: 23, 24, 27or 28. With these endoglucanases improved hydrolysis results also atmoderate and elevated temperatures with high dry matter conditions wereobtained.

In one preferred embodiment of the invention the enzyme preparationcomprises cellobiohydrolase CBHI/Cel 7A, cellobiohydrolase CBHII/Cel6A,endoglucanase EGII/Cel5A, beta-glucosidase βG/Cel3A, xylanase Xyn10A andendoglucanase fusion protein comprising an amino acid sequence havingSEQ ID NO: 24.

In another preferred embodiment of the invention the enzyme preparationcomprises cellobiohydrolase CBHI/Cel 7A, cellobiohydrolase CBHII/Cel6A,endoglucanase EGII/Cel5A, beta-glucosidase βG/Cel3A, xylanase Xyn10A andendoglucanase fusion protein comprising an amino acid sequence havingSEQ ID NO: 27.

In another preferred embodiment the enzyme preparation comprisescellobiohydrolase CBHI/Cel 7A, cellobiohydrolase CBHII/Cel6A,endoglucanase EGII/Cel5A, beta-glucosidase βG/Cel3A, xylanase Xyn10A andendoglucanase fusion protein comprising an amino acid sequence havingSEQ ID NO: 28.

As will be understood by one skilled in the art, any quantity of thecellulosic material may be used in the hydrolysis. The term “dry matter”as used herein refers to total solids, both soluble and insoluble, ofcellulosic material. The hydrolysis of cellulosic material may beconducted at low dry matter conditions, whereby by low dry matter is<15%. In other embodiments enzymatic hydrolysis may be conducted at highdry matter content, preferably >15% dry matter.

The method for treating cellulosic material with the endoglucanases ofthe invention is especially suitable for producing fermentable sugarsfrom lignocellulosic material. The fermentable sugars may then befermented by yeast into ethanol, and used as fuel. They can also be usedas intermediates or raw materials for the production of variouschemicals or building blocks for the processes of chemical industry,e.g. in so called biorefinery. The lignocellulosic material may bepretreated before the enzymatic hydrolysis to disrupt the fiberstructure of cellulosic substrates and make the cellulose fraction moreaccessible to the cellulolytic enzymes. Current pretreatments includemechanical, chemical or thermal processes and combinations thereof. Thematerial may for example be pretreated by steam explosion or acidhydrolysis.

The novel EG/Cel7 endoglucanases may be applied in any process involvingcellulolytic enzymes, such as in biofuel, biomass hydrolysis, starch,textile, detergent, pulp and paper, food, feed or beverage industry, andespecially in hydrolysing cellulosic material for the production ofbiofuel comprising ethanol. In the pulp and paper industry they may beused to modify cellulosic fibre for example in treating kraft pulp,mechanical pulp, or recycled paper.

The invention is described by the following non-limiting examples. Itwill be obvious to a person skilled in the art that, as the technologyadvances, the inventive concept can be implemented in various ways. Theinvention and its embodiments are not limited to the examples describedbut may vary within the scope of the claims.

EXAMPLES Example 1 Cloning of the Endoglucanase (cel7/egl) Genes

Standard molecular biology methods were used in the isolation and enzymetreatments of DNA (e.g. isolation of plasmid DNA, digestion of DNA toproduce DNA fragments), in E. coli transformations, sequencing etc. Thebasic methods used were either as described by the enzyme, reagent orkit manufacturer or as described in the standard molecular biologyhandbook, e. g. Sambrook and Russell (2001). Isolation of genomic DNAwas performed as described in detail by Raeder and Broda (1985).

After screening of several strains from Roal Oy culture collection onethermophilic fungal strain was selected for cloning. The probes forcloning the cel7/egl genes (egl_A and egl_B) from Acremoniumthermophilum ALKO4245 were synthesized by PCR. Degenerate oligos wereplanned basing on the alignment of the previously published amino acidsequences of GH family 7 endoglucanase (EGI) proteins. The sequences ofthe primers are shown in Table 3 (SEQ ID NOs: 1-2).

TABLE 3 The oligonucleotides used as PCR primers to amplify probes forscreening of egl genes from Acremonium thermophilum ALKO4245 Template,Oligo- SEQ genomic nucle- Length ID DNA from otides (bp) Sequence ^((a)NO: ALKO4245 egl9_s 20 TGYTGYAAYGARATGGAYAT 1 (s) ALKO4245 egl11_as 17SWRTCNARCCARTTCAT (as) 2 ^((a) N = A or G or T or C, Y = T or C, R = Aor G, S = G or C, W = A or T; “s” in the parenthesis = sense strand,“as” in the parenthesis = antisense strand.

The probes were amplified by PCR with primers described in Table 3 usingthe genomic DNA as a template in the reactions. The PCR mixtures ofAcremonium thermophilum ALKO4245 contained 1×F-511 Buffer for DynazymeDNA Polymerase (Finnzymes, Finland), 0.2 mM dNTP Mix (Fermentas,Finland), 1 μM each primer, 3% DMSO (Finnzymes, Finland) 2-4 units ofF-501L Dynazyme II DNA Polymerase (Finnzymes, Finland) and 1-2 μg of theconesponding genomic DNA. The conditions for the PCR reactions were thefollowing: 5 min initial denaturation at 95° C., followed by 28 cyclesof 1 min at 95° C., 30 sec annealing at 50° C., 30 sec extension at 72°C. and a final extension at 72° C. for 5 min.

Primer combinations described in Table 3 produced specific DNA productshaving the expected sizes (according to calculations basing on publishedcel7/egl sequences). The DNA products were isolated and purified fromthe PCR reaction mixtures and cloned into pCR4®4Blunt-TOPO® vectorsaccording to the manufacturer's instructions (Invitrogen, USA). Theinserts were characterized by sequencing and by performing Southern blothybridizations to the genomic DNA digested with several restrictionenzymes. The PCR fragments which were chosen to be used as probes forgene cloning from Acremonium thermophilum ALKO4245 strain are presentedin Table 4.

TABLE 4 The primers used in the PCR reactions, probes chosen forscreening of the egl genes from Acremonium thermophilum ALKO4245. Thegenomic template DNA and the name of the plasmid containing the probefragment are shown. Genomic DNA used as a template Fragment SEQ ForwardReverse in obtained Insert in ID Gene primer primer PCR reactions (kb)plasmid NO: egl_A egl9_s egl11_as ALKO4245 0.6 pALK2698 3 egl_B egl9_segl11_as ALKO4245 0.4 pALK2699 4

The deduced amino acid sequences from both of these PCR fragments hadsimilarity to the published EG/Cel7B sequences (BLAST program, version2.2.9 at NCBI, National Center for Biotechnology Information).

Acremonium thermophilum ALKO4245 genomic DNA was digested with severalrestriction enzymes for Southern blot analysis. The probes for thehybridization were the 566 bp (SEQ ID NO: 3 for gene egl_A) and 431 bp(SEQ ID NO: 4 for gene egl_B) EcoRI fragments, cut from the plasmidspALK2698 and pALK2699, respectively. The above probes were labeled byusing digoxigenin according to supplier's instructions (Roche, Germany).Hybridizations were performed over night at 65° C. After hybridizationthe filters were washed 2×5 min at RT using 2×SSC-0.1% SDS followed by2×15 min at 65° C. using 0.1×SSC-0.1% SDS. The E. coli strains RF8831including the plasmid pALK2698, and RF8832 including the plasmidpALK2699, were deposited to the DSM collection under the accessionnumbers DSM 25490 and DSM 25491, respectively.

From the genomic DNA of Acremonium thermophilum ALKO4245, approximate6.6 kb HindIII-digested fragment was hybridized using dioxigenin-labeled566 bp EcoRI fragment from the pALK2698 as a probe. Correspondingly,about 9.5 kb EcoRI-digested fragment was hybridized withdioxigenin-labeled 431 bp EcoRI fragment of the pALK2699 also from thegenomic DNA of the Acremonium thermophilum ALKO4245. The hybridizinggenomic DNA fragments were isolated from the pool of the digestedgenomic fragments based on their size. The genomic fragments wereisolated from agarose gel and were cloned into pBluescript II KS+(Stratagene, USA) vectors cleaved with either HindIII (gene A) or EcoRI(gene B). Ligation mixtures were transformed into Escherichia coli XL10-Gold cells (Stratagene) and plated on LB (Luria-Bertani) platescontaining 50-100 μg/ml ampicillin. The E. coli colonies were screenedfor positive clones using colonial hybridization with the pALK2698 andpALK2699 inserts as probes in the hybridization conditionscorrespondingly to that described above for Southern blot analyses.Several positive clones were collected from the plates. They were shownby restriction digestion to contain inserts of expected sizes. Thefull-length gene encoding the Acremonium thermophilum ALKO4245 EG_A(egl_A, SEQ ID NO: 5) was sequenced from the 6.6 kb HindIII insert andthe plasmid containing this insert was named pALK3152. The E. colistrain RF8939 including the plasmid pALK3152 was deposited to the DSMcollection under the accession number DSM 25492. The gene encoding theAcremonium thermophilum ALKO4245 protein A was named as egl_A.Correspondingly, the full-length gene encoding the another Acremoniumthermophilum ALKO4245 EG_B (egl_B. SEQ ID NO: 6) was sequenced from the9.5 kb EcoRI insert and the plasmid containing this insert was namedpALK3153. The E. coli strain RF8974 including the plasmid pALK3153 wasdeposited to the DSM collection under the accession number DSM 25493.The gene encoding the Acremonium thermophilum ALKO4245 protein B wasnamed as egl_B. The relevant information on the genes and the deducedprotein sequences (SEQ ID NOs: 5-8) are summarized in Table 5 and Table6, respectively.

TABLE 5 The summary on the egl genes isolated from Acremoniumthermophilum ALKO4245 Length Coding No of Lengths of with re- puta-putative SEQ introns gion tive introns ID Gene (bp) ^((a) (bp) ^((b)introns (bp) NO: egl_A 1520 1437 1 80 5 egl_B 1206 1203 0 0 6 ^((a) TheSTOP codon is included. ^((b) The STOP codon is not included.

TABLE 6 The summary of the amino acid sequences deduced from the eglgenes sequences from Acremonium thermophilum ALKO4245 Predicted MWLength (Da), Predicted EG pro- No of of ss not pI, SEQ ID tein aas SS^((a) CBM ^((b) incl ^((c) ss not incl NO: EG_A 479 18 S434 to 480104.26 7 L479 EG_B 401 24 — 39063 4.80 8 ^((a) The prediction on thesignal sequence was made using the program SignalP v3.0, NN/HMM (Nielsenet al., 1997; Nielsen & Krogh, 1998; Bendtsen et al., 2004). ^((b) Thecellulose-binding module (CBM) and linker region, the amino acids of thelinker-CBM region are indicated [M1 (Met #1) included in numbering].^((c) The predicted signal sequence was not included. The prediction wasmade using Clone Manager version 9 for Windows, Sci-Ed Software

The comparison of the deduced EG sequences from Acremonium thermophilumALKO4245 to the databases are shown in Table 7.

TABLE 7 The highest identity sequences to the deduced EG_A and EG_Bamino acid sequences from Acremonium thermophilum ALKO4245. Thefull-length amino acid sequences including the signal sequences werealigned. The database searches were performed athttp://www.ebi.ac.uk/Tools/sss/fasta/ andhttp://www.ebi.ac.uktfools/psa/emboss_needle/ using FASTA (EMBL-EBI,FASTA - Protein Similarity Search, UniProt Knowledgebase + NR PatentProteins Level-1, BLOSUM50, Gap open −10, Gap extend −2), and EMBOSSNeedle (EMBL-EBI, EMBOSS-Needle Pairwise Sequence Alignment, BLOSUM50,Gap open 10, Gap extend 0.5) for determining the degree of identity.Organism and accession number Identity (%) EG_A 100 Podospora anserina,XP_001906344.1 56.1 EG_B 100 Chaetomium globosum, EAQ91517.1 57.6 EG_A +EGI-CBM 100 Podospora anserina, XP_001906344.1 53.3 EG_B + EGI-CBM 100Trichoderma reesei, EGR48251.1 63.3 EG_A + CBHI-CBM 100 Podosporaanserina, XP_001906344.1 54.2 EG_B + CBHI-CBM 100 Thielavia terrestris,XP_003653757.1 63.0

Example 2 Production of Recombinant EG/Cel7 Proteins in Trichodermareesei

Expression plasmids were constructed for production of recombinantEG/Cel7 (EG_A and EG_B) proteins from Acremonium thermophilum ALKO4245in Trichoderma reesei. The expression plasmids constructed are listed inTable 8. The recombinant cel7/egl genes (egl_A and egl_B), includingtheir own signal sequences, were fused to the T. reesei cel7A/cbh1promoter by PCR. The transcription termination was ensured by the T.reesei cel7A/cbh1 terminator and the A. nidulans amdS marker gene wasused for selection of the transformants as described in Paloheimo et al.(2003). The linear expression cassettes (FIG. 1) were isolated from thevector backbones after NotI digestions and were transformed into T.reesei protoplasts. The host strain used does not produce any of thefour major T. reesei cellulases (CBHI, CBHII, EGI, EGII). Thetransformations were performed as in Penttilä et al. (1987) with themodifications described in Karhunen et al. (1993), selecting acetamideas a sole nitrogen source (amdS marker gene). The transformants werepurified on selection plates through single conidia prior to sporulatingthem on PD.

TABLE 8 The expression cassettes constructed to produce EG_A and EG_Brecombinant proteins from Acremonium thermophilum ALKO4245 inTrichoderma reesei. The overall structure of the expression cassetteswas as described in FIG. 1. Endoglucanase (Cel7) Expression plas-Expression protein mid cassette ^((a) EG_A pALK3156 6.9 kb NotI EG_BpALK3157 6.5 kb NotI ^((a) The expression cassette for T. reeseitransformation was isolated from the vector backbone by using NotIdigestion.

The EG/Cel7 production of the transformants was analyzed from theculture supernatants of the shake flask cultivations. The transformantswere inoculated from the PD slants to shake flasks containing 50 ml ofcomplex lactose-based cellulase inducing medium (Joutsjoki et al. 1993)buffered with 5% KH₂PO₄. The EG/Cel7 protein production of thetransformants was analyzed from the culture supernatants after growingthem for 7 days at 30° C., 250 rpm. Heterologous production ofrecombinant proteins was analyzed by SDS-PAGE with subsequent Coomassiestaining. The genotypes of the chosen transformants were confirmed byusing Southern blot analyses in which several genomic digests wereincluded and the respective expression cassette was used as a probe.

The best producing transformants were chosen to be cultivated inlaboratory scale bioreactors. The transformants were cultivated in labbioreactors at 28° C. in cellulase inducing complex medium 3-4 days withpH control 4.2±0.2 (NH₃/H₃PO₄) to obtain material for the applicationtests. The supernatants were recovered by centrifugation and filteringthrough EK filters (Pall SeitzSchenk Filtersystems GmbH, Bad Kreuznach,Germany).

Example 3 Production of the Recombinant Acremonium thermophilum ALKO4245EG+Trichoderma reesei CBM Fusion Proteins in T. reesei

The atypical linker and CBM regions of Acremonium thermophilum ALKO4245EG/Cel7_A (EG_A) were removed and the core region of the protein wasfused to linker and CBM of Trichoderma reesei EGI/Cel7B (=EGI-CBM).Acremonium thermophilum ALKO4245 EG_B was also fused to linker and CBMof Trichoderma reesei EGI/Cel7B. For that purpose, the coding sequenceof the core regions of EG_A and EG_B and the coding sequence of thelinker and the CBM of Trichoderma reesei EGI/Cel7B were synthesized byPCR using following primers:

SEQ ID NO: 9 (forward sequence for EG_A core primer)

SEQ ID NO: 10 (reverse sequence for EG_A core primer)

SEQ ID NO: 11 (forward sequence of EGI-CBM primer)

SEQ ID NO: 12 (reverse sequence of EGI-CBM primer)

SEQ ID NO: 13 (forward sequence of EG_B primer)

SEQ ID NO: 14 (reverse sequence of EG_B primer)

SEQ ID NO: 15 (forward sequence of EGI-CBM primer)

SEQ ID NO: 16 (reverse sequence of EGI-CBM primer).

The PCR reaction mixture for synthesizing the DNA sequence encoding forEG_A core contained 1× Phusion HF Reaction Buffer (Finnzymes, Finland),7.5 mM MgCl₂, 0.2 mM dNTPs, 1 μM of each primer, 3% DMSO, 4 units ofPhusion DNA Polymerase (Finnzymes, Finland), and approximately 50 ng/200μl of template DNA, containing full-length egl_A gene from Acremoniumthermophilum ALKO4245. The conditions for the PCR reaction were thefollowing: 30 sec initial denaturation at 98° C., followed by 24 cyclesof 10 sec at 98° C., 30 sec annealing at 52.5° C. (±7.5° C. gradient),30 sec extension at 72° C. and final extension at 72° C. for 7 min. Thespecific DNA fragment in PCR reaction was obtained at annealingtemperature range from 45° C. to 60° C. The compatible restriction siteswere created to the synthesized core fragment of Acremonium thermophilumALKO4245 egl_A for the fusion to Trichoderma reesei cel7/egl1 linker-CBMregion and ligation to expression vector.

The PCR reaction mixtures for synthesizing the DNA sequences encodingfor EG_B and EGI-CBMs contained 1×F-511 Buffer for Dynazyme DNAPolymerase (Finnzymes, Finland), 0.2 mM dNTP Mix (Fermentas, Finland), 1μM each primer, 3% DMSO (Finnzymes, Finland) 2-4 units of F-501LDynazyme II DNA Polymerase (Finnzymes, Finland) and approximately 50ng/200 μl of template DNA, containing the full-length egl_B gene fromAcremonium thermophilum ALKO4245 and the full-length cel7/egl1 gene fromTrichoderma reesei. The conditions for the PCR reactions were thefollowing: 5 min initial denaturation at 95° C., followed by 28 cyclesof 1 min at 95° C., 30 sec annealing at 52.5° C. (±7.5° C. gradient), 30sec extension at 72° C. and a final extension at 72° C. for 5 min. Thespecific DNA fragments in PCR reactions were obtained at annealingtemperature range from 45° C. to 60° C. The fragments created by primercombinations described above were then digested with compatiblerestriction enzymes and ligated together. The fragments amplified byPCR, primer combinations and compatible restriction enzymes aredescribed in Table 9.

TABLE 9 PCR fragments amplified from Acremonium thermophilum ALKO4245egl_A and egl_B and from Trichoderma reesei egl1 full length genesRestriction sites at SEQ the Length ID Fragment Primer pairs 5′- and 3′-ends (bp) NO: egl_A SEQ ID NO: 9 + SacII + BsmBI 1330 17 core 10 egl_BSEQ ID NO: 13 + BamHI + Sad 1272 18 core 14 egl1- SEQ ID NO: 11 +BsmBI + XhoI 281 19 CBM 12 egl1- SEQ ID NO: 15 + SacI + AgeI 212 20 CBM16

The newly created fragments were then further ligated into expressionvectors. The PCR amplified fragments in the expression plasmids wereconfirmed by sequencing (SEQ ID NO: 17 for egl_A core, SEQ ID NO: 18 foregl_B core, SEQ ID NO: 19 for egl1-CBM, SEQ ID NO: 20 for egl1-CBM, SEQID NO: 21 for egl_A+egl1-CBM and SEQ ID NO: 23 for EG_A+EGI-CBM, SEQ IDNO: 22 for egl_B+egl1-CBM and SEQ ID NO: 24 for EG_B+EGI-CBM). Thefusion genes were also further cloned into pBluescript II KS+ vector forthe patent deposition to DSM collection under following accessionnumbers: DSM 25657=E. coli strain RF10076 including the plasmid pALK3179which contains the fusion gene egl_A+egl1-CBM. DSM 25658=E. coli strainRF10077 including the plasmid pALK3180 which contains the fusion geneegl_B+egl1-CBM.

Acremonium thermophilum ALKO4245 EG_A and EG_B were also fused to linkerand CBM of Trichoderma reesei CBHI/Cel7A (=CBHI-CBM). The egl+cbh1-CBMfusion genes were designed such a way that the atypical linker and CBMregions of Acremonium thermophilum ALKO4245 egl_A would be removed andthe remaining core region will be fused to linker and CBM region ofTrichoderma reesei cel7A/cbh1 (=cbh1-CBM). Trichoderma reesei cel7A/cbh1linker and CBM regions will be fused straight to Acremonium thermophilumcel7/egl_B, since it did not contain natural CBM. The fusion genes wereordered as synthetic constructs (from GenScript, USA). The syntheticfusion genes in plasmids were confirmed by sequencing (SEQ ID NO: 25 foregl_A+cbh1-CBM, SEQ ID NO: 26 for egl_B+cbh1-CBM, SEQ ID NO: 27 forEG_A+CBHI-CBM and SEQ ID NO: 28 for EG_B+CBHI-CBM). The E. coli strainsRF9587 including the plasmid pALK3167, which contains the fusion geneegl_A+cbh1-CBM in pUC57, and RF9588 including the plasmid pALK3168 whichcontains the fusion gene egl_B+cbh1-CBM in pUC57, were deposited to theDSM collection under the accession numbers DSM 25655 and DSM 25656,respectively.

The expression plasmids were constructed for production of recombinantEG+EGI-CBM (EG_A+EGI-CBM and EG_B+EGI-CBM) and EG+CBHI-CBM(EG_A+CBHI-CBM and EG_B+CBHI-CBM) fusion proteins (SEQ ID NO: 23-24 and27-28 corresponding nucleic acid SEQ ID NO: 21-22 and 25-26). Theexpression plasmids constructed are listed in Table 10. The constructedegl_A+egl1-CBM, egl_B+egl1-CBM, egl_A+cbh1-CBM and egl_B+cbh1-CBM fusiongenes were fused to the T. reesei cbh1 (cel7A) promoter in theexpression vector. The transcription termination was ensured by the T.reesei cel7A terminator and A. nidulans amdS marker gene was used forselection of the transformants as described in Paloheimo et al. (2003).The linear expression cassettes (FIG. 1) were isolated from the vectorbackbones after NotI digestions and were transformed into T. reeseiprotoplasts. The host strain used does not produce any of the four majorT. reesei cellulases (CBHI, CBHII, EGI, EGII). The transformations wereperformed as in Penttilä et al. (1987) with the modifications describedin Karhunen et al. (1993), selecting acetamide as a sole nitrogen source(amdS marker gene). The transformants were purified on selection platesthrough single conidia prior to sporulating them on PD.

TABLE 10 The expression cassettes constructed to produce EG + CBM fusionproteins in Trichoderma reesei. The overall structure of the expressioncassettes was as described in FIG. 1. Expression plas- Expression Fusionprotein mid cassette ^((a) EG_A + EGI-CBM pALK3158 6.9 NotI EG_B +EGI-CBM pALK3159 6.7 NotI EG_A + CBHI-CBM pALK3161 6.9 NotI EG_B +CBHI-CBM pALK3162 6.7 Notl ^((a) The expression cassette for T. reeseitransformation was isolated from the vector backbone by using NotIdigestion.

The EG+CBM fusion protein production of the transformants was analyzedfrom the culture supernatant of the shake flask cultivations. Thetransformants were inoculated from the PD slants to shake flaskscontaining 50 ml of complex lactose-based cellulase inducing mediumJoutsjoki et al. 1993) buffered with 5% KH₂PO₄. The EG+CBM fusionprotein production of the transformants was analyzed from the culturesupernatants after growing them for 7 days at 30° C., 250 rpm.Heterologous production of recombinant proteins was analyzed by SDS-PAGEwith subsequent Coomassie staining. The genotypes of the chosentransformants were confirmed by using Southern blot analyses in whichseveral genomic digests were included and the respective expressioncassette was used as a probe.

The best producing transformants were chosen to be cultivated inlaboratory scale bioreactors. The transformants were cultivated in labbioreactors at 28° C. in cellulase inducing complex medium 3-4 days withpH control 4.2±0.2 (NH₃/H₃PO₄) to obtain material for the applicationtests. The supernatants were recovered by centrifugation and filteringthrough EK filters (Pall SeitzSchenk Filtersystems GmbH, Bad Kreuznach,Germany).

Example 4 Hydrolysis of Corn Fibre Substrate with Enzyme PreparationsComprising a Recombinant EG/Cel7 and EG/Cel7+CBM Endoglucanases

Steam exploded corn fibre was suspended in 0.05 M sodium citrate buffer,pH 4.8. The final weight of the hydrolysis mixture was 1 g of which thetotal solids concentration was either 5% (w/w) or 17% (w/w). Thesubstrate was hydrolyzed using different enzyme mixtures at a dosage of0.5 mg of protein per g of total solids in 2 ml reaction tubes. Theprotein contents of the enzyme components were determined using thePierce BCA assay kit (Thermo Scientific) with Bovine Serum Albumin(Thermo Scientific) as standard. The reaction tubes were agitated in alinear-shaking water bath 1086 from GFL, adjusted in differenttemperatures. For each sample point, a sample of 0.5 ml was taken fromduplicate reaction tubes, and centrifuged, the supernatant was boiledfor 20 minutes to terminate the enzymatic hydrolysis, and analyzed forreaction products from the hydrolysis. Seven separate mixturecombinations were prepared (a basis mixture MIXTURE 1, MIXTURE 1_EG_Aand MIXTURE 1_EG_B, MIXTURE 1_EG_A+EGI-CBM, MIXTURE 1_EG_B+EGI-CBM,MIXTURE 1_EG_A+CBHI-CBM and MIXTURE 1_EG_B+CBHI-CBM) with differentEG/Cel7 replacements.

A basis mixture of different cellulases was prepared using the followingcomponents:

Mesophilic EGI/Cel7B preparation containing recombinant Trichodermareesei EGI/Cel7B.

CBHI/Cel7A preparation containing recombinant Acremonium thermophilumALKO4245 CBHI/Cel7A (WO2007071818).

CBHII/Cel6A preparation containing recombinant Acremonium thermophilumALKO4245 CBHII/Cel6A (WO2011080317).

EGII/Cel5A preparation containing recombinant Thermoascus aurantiacusALKO4242 EGII/Cel5A (WO2007071818) with genetically attached CBM ofTrichoderma reesei EGII/Cel5A.

β-glucosidase preparation containing Acremonium thermophilum ALKO4245β-glucosidase/Cel3A (WO2007071818).

Xylanase preparation containing Thermoascus aurantiacus ALKO4242 Xyn10Axylanase (WO2007071818).

All cellulases were heterologously produced as monocomponents inTrichoderma reesei host strain having cellulase-free background (thegenes encoding the four major cellulases CBHI/Cel7A, CBHII/Cel6A,EGI/Cel7B and EGII/Cel5A were deleted). Crude culture supernatants wereused in the mixture. The enzyme components were combined as follows toprepare a basis mixture: cellobiohydrolase CBHI/Cel7A preparation 60%,cellobiohydrolase CBHII/Cel6A preparation 15%, endoglucanase EGII/Cel5Apreparation 10%, endoglucanase EGI/Cel7B preparation 8%, β-glucosidaseβG/Cel3A preparation 4% and xylanase Xyn10A preparation 3%. This enzymemixture was designated as MIXTURE 1.

For testing EG endoglucanase performance in the hydrolysis with MIXTURE1, 8% of the EGI/Cel7B endoglucanase component of MIXTURE 1 was replacedby:

EG/Cel7 preparation containing recombinant Acremonium thermophilumALKO4245 EG_A or

EG/Cel7 preparation containing recombinant Acremonium thermophilumALKO4245 EG_B. The mixtures containing Acremonium thermophilum ALKO4245EG/Cel7 proteins EG_A or EG_B were designated as MIXTURE 1_EG_A andMIXTURE 1_EG_B, respectively.

For testing EG+CBM fusion protein performance in the hydrolysis withMIXTURE 1, 8% of the EGI/Cel7B endoglucanase component of MIXTURE 1 wasreplaced by:

EG+CBM preparation containing recombinant Acremonium thermophilumALKO4245 EG_A genetically fused to Trichoderma reesei EGI-CBM(EG_A+EGI-CBM) or

EG+CBM preparation containing recombinant Acremonium thermophilumALKO4245 EG_B genetically fused to Trichoderma reesei EGI-CBM(EG_B+EGI-CBM) or

EG+CBM preparation containing recombinant Acremonium thermophilumALKO4245 EG_A genetically fused to Trichoderma reesei CBHI-CBM(EG_A+CBHI-CBM) or

EG+CBM preparation containing recombinant Acremonium thermophilumALKO4245 EG_B genetically fused to Trichoderma reesei CBHI-CBM(EG_B+CBHI-CBM).

The mixtures containing the fusion proteins EG_A+EGI-CBM, EG_B+EGI-CBM,EG_A+CBHI-CBM or EG_B+CBHI-CBM were designated as MIXTURE1_EG_A+EGI-CBM, MIXTURE 1_EG_B+EGI-CBM, MIXTURE 1_EG_A+CBHI-CBM andMIXTURE 1_EG_B+CBHI-CBM, respectively.

For all the mixtures the hydrolysis was performed at 37° C. and 55° C.Samples were taken from the hydrolysis after 48 h, quantified by HPLCand the concentration of glucose was determined (FIG. 2).

The results show better performance of the MIXTURE 1_EG_A and MIXTURE1_EG_B at 37° C. with high (17%) dry matter content. The amount ofglucose released from corn fibre substrate was found to increase 9% forthe MIXTURE 1_EG_A and 7% for the MIXTURE 1_EG_B compared to the MIXTURE1 (FIG. 2A). At 55° C. and with high dry matter content MIXTURE 1_EG_Awas found to increase glucose yield 34% compared to the enzyme mixMIXTURE 1 (FIG. 2B).

For the EG+EGI-CBM (EG_A+EGI-CBM and EG_B+EGI-CBM) fusion proteins theresults show better performance of the MIXTURE 1_EG_A+EGI-CBM andMIXTURE 1_EG_B+EGI-CBM at 37° C. in low (5%) dry matter conditions. Theamount of glucose released from corn fibre substrate was found toincrease 30% for the MIXTURE 1_EG_A+EGI-CBM and 35% for the MIXTURE1_EG_B+EGI-CBM compared to the MIXTURE 1 (FIG. 3A). In high dry matterconditions MIXTURE 1_EG_B+EGI-CBM mix was found to increase glucoseyield 7% at both temperatures 37° C. and 55° C. compared to the enzymemix MIXTURE 1 (FIGS. 3B and 3C).

For the EG+CBHI-CBM (EG_A+CBHI-CBM and EG_B+CBHI-CBM) fusion proteinsthe results show better performance of the MIXTURE 1_EG_A+CBHI-CBM andMIXTURE 1_EG_B+CBHI-CBM at 37° C. in both low and high dry matterconditions. The amount of glucose released from corn fibre substrate wasfound to increase 22% in low dry matter conditions and 5% in high drymatter conditions for the MIXTURE 1_EG_A+CBHI-CBM, and 8% in low drymatter conditions and 5% in high dry matter conditions for the MIXTURE1_EG_B+CBHI-CBM compared to the MIXTURE 1 (FIGS. 4A and 4B). In high drymatter conditions at 55° C. MIXTURE 1_EG_B+CBHI-CBM was found toincrease glucose yield 4% compared to the MIXTURE 1 (FIG. 4C).

REFERENCES

-   Badger, P. C. (2002) Ethanol from cellulose: a general review. In    Trends in new crops and new uses. J. Janick and A. Whipkey (eds.).    ASHS Press, Alexandria, Va., USA, pp. 17-21.-   Bailey M., Siika-aho M., Valkeajärvi A. and Penttilä M. (1993)    Hydrolytic properties of two cellulases of Trichoderma reesei    expressed in yeast. Biotehnol. Appl. Biochem 17: 65-76-   Bendtsen J. D., Nielsen H., von Heijne G. and Brunak S. (2004)    Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol.    340: 783-795.-   Coen, D. M. (2001) The polymerase chain reaction. In: Ausubel, F.    M., Brent, R., Kingston, R. E., More, D. D., Seidman, J. G.,    Smith, K. and Struhl, K. (eds.) Current protocols in molecular    biology. John Wiley & Sons. Inc., Hoboken, USA.-   Gellissen, G. (ed.) (2005) Production of recombinant proteins. Novel    microbial and eukaryotic expression systems. Wiley-VCH Verlag    Gmbh&Co. Weinheim, Germany.-   Haakana H., Miettinen-Oinonen A., Joutsjoki V., Mäntylä A., Suominen    P, and Vehmaanperä J. (2004) Cloning of cellulase genes from    Melanocarpus albomyces and their efficient expression in Trichoderma    reesei. Enz. Microbiol. Technol. 34: 159-167.-   Henrissat B. (1991) A classification of glycosyl hydrolases based on    amino acid sequence similarities. Biochem. J. 280: 309-316.-   Henrissat B. and Bairoch A. (1993) New families in the    classification of glycosyl hydrolases based on amino acid sequence    similarities. Biochem. J. 293: 781-788.-   Henrissat B. and Bairoch A. (1996). Updating the sequence-based    classification of glycosyl hydrolases. Biochem. J. 316: 695-696-   Hong J., H. Tamaki, K. Yamamoto, and Kumagai H. (2003a) Cloning of a    gene encoding a thermo-stabile endo-β-1,4-glucanase from Thermoascus    aurantiacus and its expression in yeast. Biotech. Letters 25:    657-661.-   Hong J., Tamaki H., Yamamoto K. and Kumagai H. (2003b) Cloning of a    gene encoding thermostable cellobiohydrolase from Thermoascus    aurantiacus and its expression in yeast. Appl. Microbiol.    Biotechnol. 63: 42-50.-   Joutsjoki, V. V., Torkkeli T. K. and Nevalainen K. M. H. (1993)    Transformation of Trichoderma reesei with the Hormoconis resinae    glucoamylase P (gamP) gene: production of a heterologous    glucoamylase by Trichoderma reesei. Curr. Genet. 24: 223-228.-   Karhunen T., Mäntylä A., Nevalainen K. M. H. and    Suominen P. L. (1993) High frequency one-step gene replacement in    Trichoderma reesei. I. Endoglucanase I overproduction. Mol. Gen.    Genet. 241: 515-522.-   Miettinen-Oinonen A, J Londesborough, V Joutsjoki, R Lantto, J    Vehmaanperä. 2004. Three cellulases from Melanocarpus albomyces with    application in textile industry. Enzyme Microb. Technol. 34:    332-341.-   Needleman S. and Wunsch C. (1970) A general method applicable to the    search for similarities in the amino acid sequence of two proteins.    Journal of Molecular Biology 48, 443-453.-   Nielsen H., Engelbrecht J., Brunak S. and von Heijne G. (1997)    Identification of prokaryotic and eykaryotic signal peptides and    prediction of their cleavage sites. Protein Engineering 10: 1-6.

Nielsen H and A Krogh. 1998. Prediction of signal peptides and signalanchors by a hidden Markov model. In: Proceedings of the Sixthinternational Conference of Intelligent Systems for Molecular Biology(ISMB 6), AAAI Press, Menlo Park, Calif., pp. 122-130.

-   Paloheimo M., Mäntylä A., Kallio J., and Suominen P. (2003)    High-yield production of a bacterial xylanase in the filamentous    fungus Trichoderma reesei requires a carrier polypeptide with an    intact domain structure. Appl. Env. Microbiol. 69: 7073-7082.-   Penttilä M., Nevalainen H., Rättö M., Salminen E. and    Knowles J. (1987) A versatile transformation system for the    cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155-164.-   Raeder U. and Broda P. (1985) Rapid preparation of DNA from    filamentous fungi. Lett. Appl. Microbiol. 1: 17-20.-   Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular cloning,    a laboratory manual. Cold Spring Harbor Laboratory, New York, US.-   Sambrook J. and Russell D. W. (2001) Molecular cloning, a laboratory    manual. Cold Spring Harbor Laboratory, New York, US.-   Tuohy M., Walsh J., Murray P., Claeyssens M., Cuffe M., Savage A.    and Coughan M. (2002) Kinetic parameters and mode of action of    cellobiohydrolases produced by Talaromyces emersonii. Biochem.    Biophys. Acta 1596: 366-380 (abstract).-   Visser H., Joosten V., Punt P. J., Gusakov A. V., Olson P. T.,    Joosten R., Bartels J., Visser J., Sinitsyn A. P., Emalfarb M. A.,    Verdoes J. C. and Wery J. (2011) Development of a mature fungal    technology and production platform for industrial enzymes based on a    Myceliophthora thermophila isolate, previously known as    Chrysosporium lucknowense C1. Industrial Biotechnology. June 7(3):    214-223.-   Voutilainen S., Puranen T., Siika-aho, M., Lappalainen A.,    Alapuranen M., and Kallio J., (2008) Cloning, expression, and    characterization of novel thermostable family 7 cellobiohydrolases.    Biotechnology and Bioengineering Vol. 101 Nr. 3, 515-528.-   Van Tilbeurgh, H., Loonties, F., de Bruyne, C. and    Claeyssens, M. (1988) Fluorogenic and chromogenic glycosides as    substrates and ligands of carbohydrases. Methods Enzymol. 160:45-59.

The invention claimed is:
 1. A recombinant fusion protein havingendoglucanase activity and comprising an amino acid sequence having atleast about 95% sequence identity to an amino acid sequence of SEQ IDNO: 23 or SEQ ID NO:
 27. 2. The fusion protein of claim 1, whichcomprises an endoglucanase domain which originates from Acremoniumthermophilum.
 3. The fusion protein of claim 1, comprising a cellulosebinding module (CBM) of Tricoderma reesei.
 4. The fusion protein ofclaim 3, wherein said CBM is a CBM derived from T. reesei endoglucanaseI (“EGI/Cel7B”) or T. reesei cellobiohydrolase I (“CBHI/Cel7A”).
 5. Thefusion protein of claim 3, wherein the fusion protein comprises an aminoacid sequence having SEQ ID NO: 23 or SEQ ID NO:
 27. 6. An enzymepreparation in the form of spent culture medium comprising an amino acidsequence having at least about 95% sequence identity to endoglucanase A(“EG_A”) having SEQ ID NO: 7, or endoglucanase fusion protein having SEQID NO: 23 or SEQ ID NO: 27 and at least one further enzyme selected fromthe group consisting of cellobiohydrolase, endoglucanase,beta-glucosidase, beta-glucanase, xyloglucanase, xylanase,beta-xylosidase, mannanase, beta-mannosidase, α-glucuronidase, acetylxylan esterase, α-arabinofuranosidase, α-galactosidase, pectinase, endo-or exo-α-L-arabinases, endo- and exo-galactoronase, endopectinlyase,pectate lyase, pectinesterase, phenol esterase, ligninase, ligninperoxidase, manganese-dependent peroxidase, H₂O₂-generating enzyme andlaccase with or without a mediator.
 7. The enzyme preparation of claim 6comprising cellobiohydrolase CBHI/Cel 7A, cellobiohydrolase CBHII/Cel6A,endoglucanase EGII/Cel5A, beta-glucosidase βG/Cel3A, xylanase Xyn10A andendoglucanase EG_A.
 8. The enzyme preparation of claim 6 comprisingcellobiohydrolase CBHI/Cel 7A, cellobiohydrolase CBHII/Cel6A,endoglucanase EGII/Cel5A, beta-glucosidase βG/Cel3A, xylanase Xyn10A andendoglucanase fusion protein comprising an amino acid sequence havingSEQ ID NO: 23 or SEQ ID NO:
 27. 9. A method for treating cellulosicmaterial with a fusion protein of claim 1 or an enzyme preparation ofclaim 6, wherein the method comprises the following steps: i) reactingthe cellulosic material with said fusion protein or the enzymepreparation, and ii) obtaining at least partially hydrolyzed cellulosicmaterial.
 10. The method of claim 9 wherein the at least partiallyhydrolyzed cellulosic material is obtained in processing biofuel,biomass, starch, textile, detergent, pulp and paper, food, feed orbeverages.
 11. The fusion protein of claim 1, which comprises anendoglucanase domain which originates from A. thermophilum CBS 116240.